CN103826613B

CN103826613B - Hydrophobic formulation

Info

Publication number: CN103826613B
Application number: CN201280033311.7A
Authority: CN
Inventors: R·牛
Original assignee: VAXCINE Ltd
Current assignee: VAXCINE Ltd
Priority date: 2011-05-06
Filing date: 2012-05-04
Publication date: 2016-08-24
Anticipated expiration: 2032-05-04
Also published as: EP2704690A1; WO2012152709A1; CA2835191A1; US20160136250A1; CN103826613A; RU2013154038A; GB201107629D0; US20140154315A1

Abstract

The present invention relates to material preparation in the hydrophobic solvent that generally will not dissolve described material, and the method obtaining these preparations.Particularly, the present invention relates to hydrophilic species preparation in the hydrophobic solvents such as such as oil.Also describe these preparations as the application of vaccine and the application in pharmaceutical composition.

Description

Hydrophobic formulation

The present invention relates to material preparation in the hydrophobic solvent that generally will not dissolve described material, and obtain these systems The method of agent.Particularly, the present invention relates to hydrophilic species preparation in the hydrophobic solvents such as such as oil.

The present invention is especially suitable under normal circumstances insoluble in oil or the large hydrophilic molecular of other hydrophobic solvents.

For multiple application, the such as application in pharmaceutical science, food technology or cosmetic industry, operate protein Or can bring problem during similar macromole, this is because the polarity of their hydrophilic and height limits they and fat The Degree of interaction of phase or be incorporated to the degree of fat phase.Many natural systems take lipid barrier (such as, skin, thin After birth) prevent hydrophilic molecule from contacting with internal compartment；Protein dispersibility ability in lipid carrier will be opened These macromole are introduced the new way of biosystem, thus can make dredging of lipid medium containing protein and barrier Water composition is integrated rather than is repelled by it.

The another aspect that protein is incorporated in oil to bring benefit is enzyme application in organic facies.With chemical method phase Ratio, enzymatic synthesis is just becoming more and more important, this is because its much lower energy requirement, bigger substrate and product are special Many reactions that the opposite sex, high yield and making can not chemically be catalyzed are catalyzed.Recently can be about enzyme The discovery keeping activity in organic environment opens the newest probability.Therefore, lipophilic substrate and product are related to Reaction can be effectively catalyzed, and enzyme stability is generally much higher than in aqueous environments so that it is can be used for extreme In condition much, such as high temperature.One very important aspect is, relates to the such as hydrolysis such as lipase and peptidase The reaction of enzyme can the most inversely be carried out in low water environment, therefore, it is possible to synthesize multiple industrial important compound. Another Application relates to the situation of complex reaction chain, and plurality of catalyst unit needs to keep close to each other.This is permissible It it is the situation of light-initiated redox reaction.Another possibility is that and utilize enzyme to act on organic metal substrate to cause ore deposit Change thus in oil phase, produce nano-particle in a controlled manner.Prepare preformed nano-particle stablizing in oil phase Dispersion there may also be the carrying out of beneficially some surface-catalyzed reactions.

Hydroaropic substance dispersion in oil phase rather than in an aqueous medium is also to the degeneration mediated in temperature, hydrolysis, light It is useful that the aspects such as sensitivity improve its stability.Can select to keep stream within the scope of temperature wider compared with aqueous solution The oil of body or there is more full-bodied oil, thus form the bigger protection of resistance against physical damage.In mixed phase system, Protein encapsulating in oil can limit between itself and water soluble compound to interaction harmful each other, such as oxygen Change.

The compositions of the present invention or another advantage of preparation are that they are the most anhydrous, therefore to hydrolysis-stable.They Also stable to freeze-thaw, and at high temperature there is higher stability, this is likely due to the solution folding of protein Folded and degeneration necessarily requires the existence of water.This means the water that it is expected to described compositions or preparation with hydroaropic substance Property preparation is compared has much longer storage life.It addition, because these preparations are anhydrous, during they are with pharmaceutical practice The capsule compatibility more preferable, in pharmaceutical practice, gelatin and hydroxypropyl methyl cellulose (HPMC) capsule shells can absorb Therefore moisture also soften.

The dissolving in incompatible phase of this type of material can be led to be wrapped in amphiphile epitheca and realized, described amphiphile The most compatible with material to be dissolved and continuous phase.This method in patent application WO96/014871 the most Describe, wherein, lamella formative amphiphile (such as phospholipid) is dispersed in aqueous phase, thus forms little monolithic layer capsule Bubble (SUV liposome), then mixes it with macromole, removes water by lyophilizing afterwards, is subsequently added hydrophobicity (oily) Phase.It has been proposed that adding during oil, liposome membrane mutually merges and is formed and surrounds the continuous of macromole completely The monolithic layer of extension or multi-disc tunic (amphiphile epitheca).

But, the method has bigger limitation, and (utilize that visible ray scatter is scarce this is because realize being completely dissolved Lose determine be completely dissolved) needed for amphiphile higher with the ratio of solute (w/w).In the oil such as such as oleic acid, Amphiphile/solute is than generally >=7, and triglyceride needs the amphiphile of for solute 15 to 20 times to realize making us full The dissolving of meaning, in the case of mineral oil, effectively dissolving of the macromole of high concentration is generally not capable of realizing.Due to amphiphilic This dissolubility in triglyceride and other oil phase of thing is limited, and this always gives the maximum of the solute can being contained in oil phase Amount brings bigger restriction and is typically smaller than 5mg/ml.Additionally, when these oil phases being dispersed in aqueous phase formation breast During liquid, wherein the molecule of solubilising is the most easily discharged in aqueous phase, thus have lost 30% to 70% Macromole.

It has now been found that can be macromole is encapsulated than mode more efficient way disclosed in WO96/014871 In amphiphile epitheca.This is accomplished by: first amphiphile is dissolved in organic facies (such as hexamethylene) In, the solution being then dissolved in the macromole in aqueous phase is dispersed in above-mentioned hexamethylene to form water-in-oil emulsion.Logical Crossing this mode, macromole is surrounded by the amphiphile of monolayer, rather than the multilamellar described in WO96/014871 is surrounded.No Cross, it has therefore been surprisingly found that in order to implement this method, use single amphiphile (such as S-PC) and Not, and the method only when using the particular combination of amphiphile of special ratios just effectively.Therefore, this area skill Art personnel are based purely on the teaching of WO96/014871 can not obtain the present invention.

Therefore, (it can have lamella formative or not have lamella shape to utilize the combination of amphiphile disclosed by the invention Become second nature), oil product, the easy dissolution of high-concentration of these oil products can be built with mineral oil, triglyceride or Squalene Macromole, even and if these macromole can also be retained after being distributed in aqueous medium by oil phase.Even if it is not It is the essential condition of the present invention, but macromole is incorporated to the mechanism in final oil phase and may e.g. cover reverse micelle Result in (reverse micelle).

In first aspect, the present invention provides a kind of single-phase hydrophobic formulation, and described preparation comprises the hydrophilic thing in oil phase Planting and amphipathic component, described amphipathic component is including at least docusate sodium, phospholipid and nonionic amphiphile, wherein, Described hydrophilic species part is surrounded by described amphipathic component, and the hydrophilic head group of described amphipathic component Towards described hydrophilic species, and wherein, do not have chemistry mutually between above-mentioned amphipathic component and hydrophilic species Effect, such as covalent interaction；Described preparation is characterised by, described nonionic amphiphile has: comprise The lipophilic chain of 10 to 20 carbon, and comprise the head group of 2 to 10 ethyleneoxy group or 1 to 3 hydroxyl. The molecule of described hydrophilic species is the most uniformly and stably dispersed in whole hydrophobic medium.

The definition of " nonionic amphiphile " used herein has lipophilic chain defined herein and head group Amphiphile.The lipophilic chain of described nonionic amphiphile comprises 10 to 20 carbon, preferably 12 to 18 carbon, more Preferably 14 to 16 carbon.This lipophilic chain can comprise 10,11,12,13,14,15,16,17,18,19 Or 20 carbon.Described head group comprises 2 to 10 ethyleneoxy group or 1 to 3 hydroxyl.The most described head Group comprises 4 to 8 ethyleneoxy group.This head group can comprise 2,3,4,5,6,7,8,9 or 10 Individual ethyleneoxy group.Alternately, head group can comprise 1,2 or 3 hydroxyls.Nonionic amphiphile Example include polyoxyethylene 2 cetyl ether (Brij52), polyoxyethylene 2 oleyl ether, polyoxyethylene 100 six Alkyl ether, polyoxyethylene 4 cetyl ether, polyoxyethylene 4 myristyl ether, polyoxyethylene 3 stearyl ether, poly- Oxygen ethylene 4 lauryl ether, glycolic acid ethoxylate thing lauryl ether and lauryl sorbitan or its mixture.

Suitably oil phase includes: hydrocarbon, such as non-polar oil, such as vegetable oil, including Oleum Arachidis hypogaeae semen, safflower oil, soybean oil, Oleum Gossypii semen, Semen Maydis oil, olive oil, almond oil, Oleum sesami, Oleum Cocois, Oleum Ricini, Oleum Hydnocapi semen, peach kernel oil； Isopropyl myristate mineral oil, including light paraffinic, squalane and Squalene；There is the long-chain of unsaturated fatty acid Fatty acid, such as preferably oleic acid and linoleic acid；Alcohol, particularly middle chain alcohol (such as capryl alcohol) and branched long-chain alcohol (example Such as phytol), terpenoid (such as nerol and geraniol), other alcohol (the such as tert-butyl alcohol, terpineol)；Monoglyceride, Such as glycerin mono-fatty acid ester (GMO)；Other esters, such as ethyl acetate, pentyl acetate and borneol acetate；Middle chain or Long-chain monoglyceride, diglyceride or triglyceride and mixture thereof；The halogenated analogs of any of the above material, including Halogenated oil, such as long-chain fluorocarbon and organidin three ester (such as lipidiol).Be suitable for triglyceride include by Those triglyceride that the vegetable fatty acid of fractional distillation or its mixture derive.It is, for example possible to use such as octanoic acid, the last of the ten Heavenly stems Acid and the mixture of linoleic acid triglyceride, such as Miglyol818^TM, or propylene glycol, dicaprylate and two capric acid The mixture of ester, such as Miglyol840^TM。

The phospholipid with phosphatidylcholine head group, the example of this phospholipid can be used to include phosphatidylcholine (PC) Own, LYSO-PHOSPHATIDYLCHOLINE LYSOPC (haemolysis PC), SPC, sphingomyelins, the derivant of these materials any, such as Hexadecylphosphocholine or containing the amphipathic nature polyalcohol of Phosphorylcholine and halogenation amphiphile (being such as fluorinated phospholipid). In this application, term phosphatidylcholine (PC) and lecithin are used interchangeably.The natural phosphatidyl choline being suitable for may originate from appointing What usual sources, such as eggs, particularly Semen sojae atricolor.

Orient to make amphipathic component point in the way of hydrophilic species by its head group, need introduce oil phase it Before but make the preparation method that hydrophilic species are surrounded by amphiphile after the water removal.This can be by creating biphase oil bag Aqueous emulsion realizes, wherein, non-aqueous mixable ' oil phase ' be easily by such as evaporation or lyophilizing and remove hydrophobic Phase.As the method removing oil phase, lyophilizing is favourable, this is because the aqueous phase in emulsion microdroplet can be removed simultaneously.

Extremely preferred, the preparation of the present invention is optically transparent, and this can be by measuring turbid in visible light wave strong point Degree is monitored, and in some cases, by checking that the precipitation status in a period of time is monitored.Generally can measure The optical density of 620mm.0.2 value below, preferably 0.15 value below are considered as transparent.

In all structures of the present invention, the center of the hydrophilic head of amphiphile molecules inwardly structure, and hydrophobic Property afterbody is then outwardly directed to be dispersed with the solvent of hydrophobicity species.

Therefore, in a second aspect of the present invention, it is provided that a kind of manufacture contains the side of the hydrophobic formulation of hydrophilic species Method, said method comprising the steps of:

(i) by the solution of the docusate sodium being dissolved in hydrophobic solvent, phospholipid and nonionic amphiphile be dissolved in water The solution of the hydrophilic species of phase mixes to form emulsion；

(ii) described aqueous phase and hydrophobic solvent are removed；

(iii) in the residue being dried obtained in (ii), oil phase is added.

Term used herein " hydrophobic solvent " refers to the hydrophobic phase removed easily by such as evaporation or lyophilizing.Can To use any non-volatile hydrophobic solvent with applicable fusing point.This solvent must be that water is immiscible, and should When easy lyophilizing, therefore it preferably has the freezing point of-10 DEG C to+15 DEG C.The example of solvent being suitable for include hexamethylene, Cycloheptane and cyclooctane, the mixture of these compounds of multiple ratio, and with the tert-butyl alcohol of the small scale added Mixture, these solvents be enough to the dissolubility of amphiphile is brought up to desired level.The selection of hydrophobic solvent is depended on The type of species to be dissolved and amphiphile.Those skilled in the art utilize the guidance seen in following example to hold Change places and determine this selection.

It is used alone phospholipid in this procedure and will not produce the transparent dispersion liquid in various different oil.If using non- Ion-type amphiphile and the combination of docusate sodium, in the case of being completely absent free water in systems, its result is carefully Changing between suspension or the highly uniform but extremely muddy dispersion liquid of granule, this depends on the ratio of amphiphilic species Example.But, contain phospholipids as less component and but obtained transparent with docusate sodium and the combination of nonionic amphiphile Dispersion liquid.In the case of oil phase is mineral oil and nonionic amphiphile is POE2 cetyl ether (Brij52), At components by weight docusate sodium: phospholipid: nonionic amphiphile is to obtain optimum during 2:1:2 to 3:2:3.This A little ratios simply mean to show sex ratio, particular it should be pointed that definite ratio depends on the property of used oil and amphiphile Matter.As described in embodiment as forth below in this specification, easily carry out experiment any to pledging love to determine The optimal proportion of different component under condition.

The aqueous phase being suitable for includes water, deuterium oxide and dimethyl sulfoxide (DMSO).Can be by a small amount of extra hydrophilic Reagent mixes with aqueous favoring, such as ethylene glycol, glycerol, propylene glycol, Allyl carbonate, PEG or monosaccharide or widow Sugar.

The level that amphiphile is at most 100mg/ml with the total soluble matters in solvent is dissolved in hydrophobic solvent.Macromole, Preferably immunogen or immunomodulator, generally with the concentration of 10mg/ml to 20mg/ml be dissolved in water or other be suitable for In aqueous phase (such as DMSO).Then aqueous phase is joined in hydrophobic solvent, preferably with the ratio of 1:4 (volume/volume) Example, thus after vortex mixed, obtain uniform dispersion liquid.

The mean diameter of emulsion particle depends on the definite character of hydrophobic phase and aqueous phase.But, it can be that 2 μm are left Right.

Aqueous phase dispersion in hydrophobic solvent can be realized by mixing, such as by the short time (such as from about 10 to 60 Second, normally about 15 seconds) violent vortex mixed realize, or (such as use orbit determination by the gentle mixing of a few hours Shaking table) realize.

The product of the method for second aspect is novel, comprises be dissolved in such as mineral this is because it allows to preparation The combination of the hydrophilic species being generally insoluble in hydrophobic solvent in the oil such as oil, squalane, Squalene and triglyceride Thing, wherein, after described compositions being dispersed in aqueous phase, described hydrophilic species can be retained in higher degree In described hydrophobic solvent.Other the most normally solid oil (such as tristearins, March can also be used Osmanthus essence, paraffin), as long as carrying out oil is added the step to aqueous favoring at a temperature of the fusing point higher than involved oil Rapid.

Above-mentioned composition can be used for preparing two-phase compositions.Therefore, in the third aspect, the present invention provides one to comprise parent Aqueous phase and the two-phase compositions of hydrophobic phase, wherein, described hydrophobic phase comprises above-mentioned composition or preparation.On can be by making State compositions or hydrophobic formulation to contact with aqueous favorings such as such as aqueous solutions and form described two-phase compositions.The parent being suitable for Aqueous phase comprises water, deuterium oxide and dimethyl sulfoxide (DMSO).Can be by a small amount of extra hydrophilic agent with hydrophilic Mix mutually, such as ethylene glycol, glycerol, propylene glycol, Allyl carbonate, PEG or monosaccharide or oligosaccharide.

In the preferred implementation of the third aspect, two-phase compositions is O/w emulsion.Hydrophobicity containing the present invention The emulsion of preparation or compositions can be used for preparing microcapsule.If the emulsion aqueous phase containing gelatin is formed, then may be used To make gelatin separate out from solution with known method by cohesion, and can be outside the microdroplet containing the hydrophobic phase of hydrophile Enclose formation film.After removing aqueous favoring, microcapsule will retain.This technology is as known in the art, but with this Bright preparation is particularly useful when combining.Therefore, a fourth aspect of the present invention provides the method preparing O/w emulsion, Said method comprising the steps of: make the single-phase hydrophobic formulation of the present invention contact with aqueous favoring to form oil-in-water breast Liquid.

In a preferred embodiment, aqueous favoring comprises gelatin or albumin.

Hydrophilic solutes has been retained in hydrophobic oil phase by the double emulsion of this oil-in-water, and with different time to external aqueous In region, the degree of seepage is minimum.The oil used in this system is preferably mineral oil, squalane, Squalene or glycerol three Ester.Other the most normally solid oil (such as tristearin, trilaurin, paraffin) can also be used, only To carry out oil is added the step to aqueous favoring at a temperature of the fusing point higher than involved oil.

If external hydrophilic is albumin (such as concentration is the albumin of 50mg/ml) or at most 20%w/w level mutually Gelatin, then can further enhance stick effect.Therefore aqueous favoring preferably comprises gelatin or albumin.Reserving degree is more Height, the most described goods are more suitable as vaccine delivery vector.

The product of the present invention has extremely many purposes, and has multiple application.At the 5th aspect, the present invention provides a kind of system Product, described goods comprise preparation or the compositions of the present invention, and optionally one or more medicines can accept excipient, Diluent or supporting agent.Described goods can be used for medical applications.

In a preferred embodiment of the present invention, the hydrophilic species in described compositions or preparation are immunogens. Described goods are preferably vaccine.On the other hand, the invention provides the goods application as vaccine of the present invention.

In the present invention, term " hydrophilic species " relates to being usually soluble in aqueous solvent but insoluble in hydrophobic solvent Any species.The wide range of the hydrophilic species in the present invention, but large hydrophilic molecular represents and can use The example of species.

Multiple macromole is suitable for the present invention.Generally, this macromolecular compound has hydrophilic or at least has parent Aqueous areas, seldom has any problem this is because hydrophobic macromolecules is dissolved in oily solution.The macromole being suitable for Example include protein and glycoprotein, few nucleic acid and Polynucleotide (such as DNA and RNA), polysaccharide and any this The super-molecule assembling body (including whole cell or organelle in some cases) of a little materials.The method of the available present invention becomes The example of the concrete albumen that merit is dissolved includes insulin, calcitonin, hemoglobin, cytochrome C, Radix Cochleariae officinalis peroxidating Thing enzyme, aprotinin, Mushroom Tyrosinase, erythropoietin, auxin, growth hormone, hormone releasing factor Son, Garland peptide, urokinase, factors IX, tissue-type plasminogen activator, superoxide dismutase, peroxidating (all above-mentioned albumen all may be from appointing for hydrogen enzyme, peroxidase, ferritin, interferon, Factor IX and fragment thereof The source what is suitable for).The glucosan that other macromole are FITC labelling that can use and the RNA of Torulla yeast Extract.Especially, macromole can be collagen, such as type i collagen or II Collagen Type VI.Containing II Collagen Type VI Goods are the immunoreactive the most promising material standed fors in oral downward rheumatoid arthritis.Macromole also may be used To be immunogen, it is particularly useful for vaccine combination.Generally introducing least concentration in the oil of 1ml is the big of 2.5mg Molecule, therefore the macromole concentration in initial hydrophilic solvent is at least 10mg/ml.

Term used herein " immunogen " relates to cause the species of immune result.This result can be typically to exempt from Epidemic disease response, such as, produce antibody or trigger differentiation or the amplification of specific T cells group, and can be systematic or office Portion, such as it is limited to mucosa response.Alternately, immune result can also is that such as immunologic tolerance, wherein makes Primary immune system excites nonreply to specific antigen.Selecting as another, described result can be desensitization, The trend that the autoimmune for specific antigen or anaphylaxis (IgE) occur wherein being pre-existing in reduces.

Immunogen be selected from but be not limited to diphtheria toxoid, tetanus toxoid, Clostridium botulinum toxoid, venom antigen, Virus antigen (the such as antigen of first, second, third, fourth or hepatitis E virus, pertussis subunit, influenza A and/or Influenza B virus (full inactivation of viruses or protein protomer), H1N1 swine influenza virus, H5N1 bird flu virus, spinal cord Poliovirus, rotavirus, mumps virus, Measles virus, chickenpox virus, meningitis virus, rubella virus, Respiratory syncytial virus, HIV, EV71, Dengue Virus Antigen, yellow fever virus antigen, human papillomavirus Antigen, herpesvirus HSV1 or HSV2 antigen, Ebola virus, porcine reproductive and respiratory syndrome virus, pig ring Shape virus 2 types, west Nile virus, Japanese encephalitis virus, hand-foot-mouth disease antigen), whole antibacterial or its extract (example Such as BCG, other antigen of mycobacterium, intestinal tract disease pathogen and antigen thereof, including such as cholera antigen, sramana Ella species, Escherichiaspp, Heliobacter pylori antigen, Pseudomonas aeruginosa, Chiamydia species, Neisser ball Ella species, yersinia species), fungus or fungal antigen, A type or H influenzae type B (with or not With carrier protein), protozooal antigens (such as malaria, leishmania, toxoplasma, trypanosomicide), trematodiasis antigen (example Such as schistosomicide), cestode antigen (such as, from cysticercus, echinococcus), nematicide antigen (such as bend ascarid, ancylostome and Filaricide), spiral belong to the special skin covering of the surface epi-position of antigen (such as Borrelia species), cancerous cell and targeted cells receptor Antiinflammatory regulatory factor, the polymer conjugate of steroid.Include that HLA resists for lowering the immunogen of immunne response Former, pollen, house dust mite antigen, bee sting or food allergen, such as seitan or Semen arachidis hypogaeae, glutamte dehydrogenase, it is used for controlling Treat the insulin of diabetes or the conjugate containing insulin subfraction.It addition, immunogen can be collagen, such as I Collagen Type VI or II Collagen Type VI.Goods containing II Collagen Type VI are the immunity in oral downward rheumatoid arthritis The most promising material standed for of reaction.Immunogen can be peptide, protein, lipid, sugar, nucleic acid, steroid and/ Or the conjugate that the combination of one or more these reagent is formed.When antigen is peptide, polysaccharide or other antigen, also may be used So that its with at least one in chain or long chain hydrocarbon suffix close.

An advantage of the invention that different antigen (such as albumen and polysaccharide) can coexist in identical carrier, from And caused the immunne response of enhancing as the supporting agent of another kind of component by a kind of component, and without any covalency even Connect.

In the case of needs raise immunne response, immunogen can be with one or more other molecule (immunostimulant Or adjuvant) combine, jointly embed in same oil phase together as immunogen.Described adjuvant comprises the steps that cholera toxin B Fragment and the like and derivant, HLT and the like and derivant, BCG, contains The oligonucleotide sequence of CpG, tetanus toxoid, diphtheria toxoid, bacteria lipid A (intact or detoxification), single Monophosphoryl lipid A.

In another embodiment, immunogen and one or more cytokines dissolve to strengthen response altogether.Be suitable for is thin The example of intracellular cytokine includes IL-4, IL-10, IL-12 and IFN-γ.Other immunostimulant, example can also be added Such as monophosphoryl lipid A, Mycobacterium extract, muramyldipeptide and the like, tuftsin and cholera subunit B and colibacillary heat-labile toxin.

Can also easily by little molecule (such as vitamin) with macromole (particularly polysaccharide, such as cyclodextrin) together altogether Dissolve.Little molecule (such as vitamin B12) can also be with macromolecular chemistry coupling, such that it is able to comprise in the composition.

Compared with the method for the prior aries such as such as WO95/13795, the method for the present invention can be with much lower amphiphilic Thing: protein ratio is encapsulated.This can make more macromole be incorporated in the such as oil such as triglyceride and mineral oil. It addition, after being distributed in aqueous medium, macromole reservation in oil is higher.

In addition to macromole, the method for the present invention can be used for dissolving less organic molecule.The example of organic molecule Including glucose, CF 5(6)-Carboxyfluorescein and various medicaments, such as anticarcinogen, but described method is equally applicable to it certainly His organic molecule, such as vitamin or forms of pharmacologically active agents or biologically active agent.It addition, utilize the method all right Dissolve the such as compound such as calcium chloride and sodium phosphate.It practice, the present invention has especially for pharmacy or biologically active agent Profit, this is because use non-aqueous solution that molecule can be made to enter internal path variation, thus such as increases biology Availability.

The another type of species that can be included in the hydrophobic composition of the present invention are inorganic material, the most inorganic little point Son or colloidal substance, such as colloidal metal.The method of the present invention can make colloidal metal (such as gold colloidal, palladium, platinum Or rhodium) even if properties also be able to be protected in the hydrophobic solvent that can make particle aggregation under normal circumstances Stay.The catalysis of this reaction for carrying out in organic solvent can be particularly useful.

Other bulky grain materials can also utilize the method to encapsulate, such as virus and antibacterial (either live, reduction Or inactivation).

In other respects, the present invention provides:

A kind of cosmetic preparation, preparation or compositions that described goods comprise the present invention and optionally one or more are composed Shape agent, diluent or supporting agent.

A kind of method treating study subject, described method includes preparation or the compositions using the present invention.

Comprise preparation or the compositions of the present invention of collagen or its fragment, be used for treating rheumatoid arthritis.Described glue Former preferably II Collagen Type VI.

Can be by thing insoluble in lipophilic solvent under normal circumstances to use a kind of mode of compositions of the present invention Matter oral delivery gives mammal including people.This may be used for delivering dietary supplement, such as vitamin, or For delivering bioactive substance, particularly protein or glycoprotein, including insulin, growth hormone and immunogen.

In another kind is applied, by such as said method, the nutrients such as such as vitamin can be encapsulated into capsule or micro- In capsule, subsequently, it serves not only as human foods supplement, it is also possible to for agricultural and aquatic products industry, the latter's One example is for cultivating the production of the foodstuff of juvenile prawn.

It addition, described compositions can be used for preparing the medicine of parenteral administration or other goods and making for local or eye Goods.This is applied, it is usually preferred to use above-mentioned oil solution and the emulsion of aqueous phase.

Many treatments or Prevention Processing realize sustained release or postpone release through being designed to, or relate to bi-component body System, described bicomponent system includes the most i.e. releasing component and for postponing release or the component of sustained release.Due to it High stability, the preparation of the present invention has especially for the macromole goods being designed to carry out sustained release or delay release With.

The longer storage life of the present composition is advantageous particularly in drug world.

This oil bag hydrophile preparation can be used for covering abnormal smells from the patient in pharmacy or similar industry.This is in pharmaceutical industries One special problem, many medicines are because having offensive odour and not joyous by patient, particularly child Meet.

Another Application is in cosmetic industry, wherein, the hydrophobic formulation of hydrophilic compounds is incorporated to cosmetic preparation In the most very easy.The macromole that can use by this way includes that those have certain wetting action or enzymatic The macromole of effect.The present invention can be also used for being incorporated in dermatosis emulsifiable paste and lotion the protein such as such as collagen.

The goods of the present invention can exist with other agents, so that it can be applied to humans and animals to realize controlling Treat purpose or other purposes.The compositions of gained can include that paste, emulsifiable paste, gel, semi-solid or biphase solid divide A prose style free from parallelism.Described goods can local application, Orally administered, ocular administration, nasal administration or use as suppository, or can (such as intramuscular injection, subcutaneous injection or intradermal injection) is used as injection.Carry out Orally administered in the case of, Described compositions can be liquid, lozenge, gel form or mix with dried powder, and these forms any are all with freedom Form is absorbed or to be encapsulated in such as gelatin, starch or HPMC hard capsule case or soft capsule (such as Perle) In form picked-up.Optionally, these capsules can have enteric coating and do not hold with gastric to pass them through stomach Thing interacts, but dissolves in small intestinal or large intestine subsequently.The coating being suitable for is known in the art.As another Selecting, for applicable route of administration, such as nasal administration, pulmonary administration, buccal are used and sublingual administration, combination Thing can be aerosol form.Aerosol can be by the oily microdroplet of the hydrophobic formulation containing the application or oil-in-water microdroplet Formed.

The compositions of the present invention is suitably adapted for being used by any suitable approach, such as, (include buccal by oral And Sublingual) approach, anal route, nasal, the locally approach that (includes buccal, Sublingual or transdermal), vaginal approach or Parenteral (including subcutaneous, intramuscular, intravenous or Intradermal) approach.Described goods can be by known in pharmaceutical field Prepared by any method, such as by being combined with supporting agent or excipient by active component.

It is suitable for Orally administered pharmaceutical preparation to exist as discrete unit, such as capsule or tablet；Powder or Granule；Solution in aqueous or non-aqueous liquid or suspension；Edible foam or stirring thing (whip)；Emulsion.

Be suitable for the pharmaceutical preparation of local application can be configured to ointment, emulsifiable paste, suspension, lotion, powder, solution, Paste, gel, spray, aerosol or oil.

For using to eye or other outside organization (such as mouth and skin), described goods are preferably as topical ointment Or emulsifiable paste uses.When being configured to ointment, can be by active component and paraffin substrate or the mixable ointment base of water one Rise and use.Alternately, by Oil-in-water emulsifiable paste substrate or Water-In-Oil substrate, active component can be configured to breast Cream.

Being suitable for the pharmaceutical preparation of eye local application is included eye drop, wherein, active component is dissolved in or is suspended in suitable In the supporting agent (particularly aqueous solvent) closed.

It is suitable for the pharmaceutical preparation of local application in mouth and includes lozenge, pastille and mouth wass.

It is suitable for the pharmaceutical preparation to rectal administration to exist as suppository or enema.

Supporting agent is that the pharmaceutical preparation being suitable for nasal administration of solid includes that particle diameter is the thick of such as 20 μm to 500 μm Powder, it is used in the way of smelling, i.e. quickly sucked by nasal meatus from lifting nose in powder container nearby. Supporting agent is the microemulsion that the applicable goods used as nasal spray or nasal drop of liquid include comprising active component Aqueous solution or oil solution.

Being suitable for sucking the pharmaceutical preparation used and include fine ash or smog, it can use various types of metering Medicine-feeding type to add Calm the anger colloidal sol, aerosol apparatus or insufflator produces.

Be suitable for vaginal application pharmaceutical preparation can as pessary, tampon, emulsifiable paste, gel, paste, foam or Spray product exists.

The pharmaceutical preparation being suitable for parenteral administration includes: aqueous and nonaqueous aseptic parenteral solution, and it can contain antioxygen Agent, buffer agent, antibacterial and make the isotonic solute of the blood of receiver of these goods and plan；Aqueous and non-aqueous Sterile suspensions, it can comprise suspending agent and thickening agent.These goods may reside in unit dose or multiple dose holds In device, the most airtight ampere bottle and bottle, and under the conditions of lyophilization (lyophilizing) can be stored in, only need in sight Sterile liquid carriers (such as water for injection) will be added during use.I.e. can use aseptic powder with injection solution and suspension Prepared by end, granule and tablet.

It should be appreciated that in addition to the most specifically mentioned composition, described goods are further included in involved Other reagent conventional use of in the association area of product type, such as, are suitable for Orally administered goods and can comprise tune Taste agent.

Finally, the present invention has multiple application in chemistry and biosynthesis field (the most anhydrous enzyme' s catalysis).

The present invention is further described below with reference to following non-limiting example and the following drawings:

Fig. 1 shows the sickness rate of mouse arthritis after the preparation for treating by the present invention comprising collagen.

Fig. 2 shows the preparation using the present invention the comprising collagen effect to arthritic mice cartilage erosion.

Embodiment 1

The combination utilizing docusate sodium, Brij52 and SPC forms the reverse micelle containing aprotinin in mineral oil

1. weigh in the vial of 8ml band screw lid 300mg docusate sodium, and add 2.7ml hexamethylene. After being tightened by lid, vial shaken of heating is until contents melting, thus obtains the solution of concentration about 100mg/ml.

2. weigh in the vial of 8ml band screw lid 300mg Brij52 (polyoxyethylene 2 cetyl ether), And add 2.7ml hexamethylene.After being tightened by lid, vial shaken of heating is until contents melting, thus obtains concentration The solution of about 100mg/ml.

3. weigh in the vial of 8ml band screw lid 300mg S-PC, and add 2.7ml Hexamethylene.After being tightened by lid, vial shaken of heating is until contents melting, thus obtains concentration about 100mg/ml Solution.

4. the vial of 10 2ml band screw lids is labeled as 1 to 10, each pipe distributes volume shown in following table The solution from step 1,2 and 3.

Pipe is numbered	Docusate sodium solution	Brij52 solution	S-PC
					μl	μl	μl
1	0	400	-
				2	100	300	-
3	200	200	-
				4	300	100	-
5	400	0	-
				6	0	400	100
7	100	300	100
				8	200	200	100
9	300	100	100
				10	400	0	100

The aprotinin solution of 5 20mg/ml adding 100 μ l in each sample, of short duration vortex mixed, then sweet Quick freezing in oil bath, is subsequently exposed in the vacuum of below 1mbar overnight, to remove hexamethylene and water.

In each bottle, within 6 second days, add 450 μ l mineral oil, subsequently bottle capping be placed on roll mixer, Until obtain single homogeneously.

In the 7 single holes that each dispersion liquid of 200 μ l is transferred in microwell plate, by surveying on microtiter plate reader Optical density at amount 620nm evaluates scattering.

From following table, when the combination using two or more amphiphiles forms hydrophobic phase time, it was observed that maximally effective Dissolve (judging according to the scattering disappearance indicated by OD minimizing).

Sample without PC	Absorbance reading	Sample containing PC	Absorbance reading
				1	0.514	6	1.041
2	0.34	7	1.22
				3	0.2	8	0.089
4	0.231	9	0.076
				5	0.928	10	0.49

Embodiment 2

When being applied in combination docusate sodium, Brij52 and SPC, in aprotinin reverse micelle from mineral oil Leakage scenarios

1. in 2ml bottle, add PBS and the aprotinin oil product of 20 μ l embodiments 1 of 400 μ l.By sample It is vigorously mixed to dispersion, then in centrifuge, is centrifuged 10 minutes with 3000rpm.

2., in the single hole being transferred in microwell plate by 50 μ l aqueous phases of each sample, it is added thereto to 150 μ l Bradford protein reagent.Protein concentration is determined by measuring the optical density at 570nm on microtiter plate reader, And compare with standard curve.By with contain only comparing of aprotinin and infer leakage scenarios.

From following table, when the combination using two or more amphiphiles forms hydrophobic phase time, and seepage is minimum.

Sample without PC	% seepage	Sample containing PC	% seepage
				1	12.42	6	1.86
2	0.38	7	0.90
				3	0.27	8	0.39
4	0.27	9	0.25
				5	0.81	10	0.42

Embodiment 3

The combination utilizing docusate sodium, Brij52 and SPC is formed in mineral oil and contaminates containing macromolecule polyalcohol Expect the reverse micelle of poly-R-478

1 weighs 300mg docusate sodium in the vial of 8ml band screw lid, and adds 2.7ml hexamethylene. After being tightened by lid, vial shaken of heating is until contents melting, thus obtains the solution of concentration about 100mg/ml.

2 weigh 300mg Brij52 (polyoxyethylene 2 cetyl ether) in the vial of 8ml band screw lid, And add 2.7ml hexamethylene.After being tightened by lid, vial shaken of heating is until contents melting, thus obtains concentration The solution of about 100mg/ml.

3 weigh 300mg S-PC in the vial of 8ml band screw lid, and add 2.7ml Hexamethylene.After being tightened by lid, vial shaken of heating is until contents melting, thus obtains concentration about 100mg/ml Solution.

The vial of 10 2ml band screw lids is labeled as 1 to 10 by 4, distributes volume shown in following table in each pipe The solution from step 1,2 and 3.

The poly-R-478 solution of 5 20mg/ml adding 100 μ l in each sample, of short duration vortex mixed, then exist Quick freezing in glycerol bath, is subsequently exposed in the vacuum of below 1mbar overnight, to remove hexamethylene and water.

In the 7 single holes that each dispersion liquid of 200 μ l is transferred in microwell plate, by surveying on microtiter plate reader Optical density difference between amount 620nm and 492nm evaluates scattering.

Sample without PC	Absorbance reading	Sample containing PC	Absorbance reading
				1	0.87	6	0.129
2	0.27	7	0.043
				3	0.208	8	0.078
4	0.485	9	0.147
				5	0.561	10	0.416

Embodiment 4

When being applied in combination docusate sodium, Brij52 and SPC, the poly-R-478 of macromole dyestuff is from mineral oil Reverse micelle in leakage scenarios

1 PBS adding 400 μ l in 2ml bottle and the poly-R-478 oil product of 20 μ l embodiments 1.By sample Product are vigorously mixed to dispersion, are then centrifuged 10 minutes with 3000rpm in centrifuge.

In the 2 single holes that the aqueous phase of 200 μ l of each sample is transferred in microwell plate, by microtiter plate reader Optical density at upper measurement 492nm determines dye strength, and compares with standard curve.By with contain only poly- Leakage scenarios is inferred in comparing of R-478.

Sample without PC	% seepage	Sample containing PC	% seepage
				1	100	6	15
2	32	7	4
				3	25	8	8
4	59	9	17
				5	68	10	50

Embodiment 5

Compared with the situation only using S-PC, it is being applied in combination docusate sodium, Brij52 and Semen sojae atricolor phosphorus Leakage scenarios in lysozyme reverse micelle from mineral oil during phosphatidylcholine

Constructed as below utilize S-PC as the hydrophobic formulation of amphiphile, then add oil phase:

1. S-PC (SPC) is added in the distilled water in 20ml bottle (1g SPC+9ml water), then By mixture vortex mixed until being completely dispersed.

Dispersion liquid is pressed through 0.2um Anatop filter twice the most subsequently.

3. in a 8ml bottle, weigh 20mg lysozyme and be dissolved in the 4ml lipid from above step 2 In dispersion liquid.

4. the lysozyme soln from step 3 of 6 part of 400 μ l aliquot is transferred to new 2ml band screw lid Vial in, then quick freezing in glycerol, and at 30 DEG C keep 1 hour.

The most then by bottle lyophilizing overnight.

Constructed as below utilize docusate sodium, Brij52 and S-PC as the hydrophobic formulation of amphiphile, so Rear interpolation oil phase:

1. S-PC (SPC) is dissolved in (600mg in the hexamethylene in 8ml bottle with the concentration of 100mg/ml SPC+5.4ml hexamethylene).

2. docusate sodium is dissolved in (600mg docusate sodium in the hexamethylene in 8ml bottle with the concentration of 100mg/ml + 5.4ml hexamethylene).

3. Brij52 is dissolved in (600mg Brij52+5.4ml in the hexamethylene in 8ml bottle with the concentration of 100mg/ml Hexamethylene).

4. lysozyme is dissolved in the distilled water in 8ml bottle with 20mg/ml.

5. in 20ml bottle, be separately added into above each solution of 2ml, 3ml and 3ml and mix, thus with than Example (2:3:3) mixing SPC, docusate sodium and Brij52 solution.

6. in the vial of 6 2ml band screw lids, add SPC:Brij52: the docusate sodium/hexamethylene of 400 μ l Solution.

7. in each bottle obtained in previous step, add 100 μ l lysozyme solns and carry out vortex mixed (about simultaneously 10 seconds).Freezing dispersion liquid, incubation about 1 hour at 30 DEG C subsequently in the glycerol bath of 20 DEG C immediately.

By sample lyophilizing overnight the most subsequently.

Oil phase is prepared as follows from dried residue achieved above:

9., when sample drying, in each bottle, add the different oil set forth below of 360 μ l.Subsequently bottle is sealed Lid is placed on roll mixer, until obtaining single uniform oil phase.

Sample	Added oil
		1	Mineral oil
2	Squalene
		3	Glycerin mono-fatty acid ester
4	Miglyol^TM840
		5	Miglyol^TM818
6	Medium chain mono

In the following manner albumen seepage from hydrophobic phase after being dispersed in aqueous phase is carried out quantitatively:

1. glimmering amine is dissolved in acetone (2mg glimmering amine+10ml acetone) with 0.2mg/ml.

2. in the bottle of 12 8ml tape labels, add PBS and each hydrophobic phase of 75 μ l of 1.5ml. The most acutely shake sample, and with 1000g centrifugal sedimentation 50 minutes.

The most then 1ml is transferred in new bottle from the aqueous phase of each sample, with the glimmering amine/acetone soln of 50 μ l Vortex mixed 10 seconds together.

4. every part of sample of 200 μ l is transferred on white microwell plate, at Molecular Devices Spectramax With Ex=390nm on fluorescent plate reader;Em=465mn;Intercepting value=455nm measures fluorescence.

5. the free lysozyme utilizing multiple concentration prepares standard curve, and calculates the percentage accounting for introduced original vol It is used for albumen leakage values from each oil phase.

From this table, for multiple different oil, different, when using amphiphile from when only using phosphatidylcholine Combination time protein seepage from oil greatly reduce.

Embodiment 6

Multiple different amphiphile is utilized to be formed in mineral oil instead with the combination of docusate sodium and S-PC Micelle

The 3 every kind of amphiphiles shown in following table weighing 300mg in the vial of 8ml band screw lid, and add 2.7ml hexamethylene.After being tightened by lid, vial shaken of heating is until contents melting, thus obtains concentration about 100mg/ml Solution.

4 weigh 300mg S-PC in the vial of 8ml band screw lid, and add 2.7ml Hexamethylene.After being tightened by lid, vial shaken of heating is until contents melting, thus obtains concentration about 100mg/ml Solution.

The vial of 10 2ml band screw lids is labeled as 1 to 10 by 5, distributes volume shown in following table in each pipe The solution from step 1,2 and 3.

Pipe is numbered	Docusate sodium solution	Amphiphile solution	S-PC
					μl	μl	μl
1	0	400	-
				2	100	300	-
3	200	200	-
				4	300	100	-
5	400	0	-
				6	0	400	100
7	100	300	100
				8	200	200	100
9	300	100	100
				10	400	0	100

The lysozyme soln of 5 20mg/ml adding 100 μ l in each sample, of short duration vortex mixed, then sweet Quick freezing in oil bath, is subsequently exposed in the vacuum of below 1mbar overnight, to remove hexamethylene and water.

Polyoxyethylene 10 cetyl ether

Sample number into spectrum	Without PC	Sample number into spectrum	Containing PC
				1	2.179	6	2.148
2	1.377	7	1.09
				3	0.142	8	0.156
4	0.08	9	0.091
				5	0.359	10	0.101

Polyoxyethylene 2 stearyl ether

Sample number into spectrum	Without PC	Sample number into spectrum	Containing PC
				1	1.693	6	1.716
2	0.124	7	0.105
				3	0.096	8	0.099
4	0.085	9	0.094
				5	0.462	10	0.101

Polyoxyethylene 4 cetyl ether

Sample number into spectrum	Without PC	Sample number into spectrum	Containing PC
				1	0.604	6	0.693
2	0.096	7	0.085
				3	0.088	8	0.096
4	0.087	9	0.084
				5	0.482	10	0.403

Polyoxyethylene 4 myristyl ether

Sample number into spectrum	Without PC	Sample number into spectrum	Containing PC
				1	0.76	6	0.546
2	0.099	7	0.091
				3	0.104	8	0.102
4	0.082	9	0.086
				5	0.378	10	0.106

Polyoxyethylene 3 stearyl ether

Sample number into spectrum	Without PC	Sample number into spectrum	Containing PC
				1	2.011	6	2.476
2	1.665	7	1.474
				3	0.093	8	0.474
4	0.081	9	0.087
				5	0.466	10	0.097

Polyoxyethylene 4 lauryl ether

Sample number into spectrum	Without PC	Sample number into spectrum	Containing PC
				1	0.401	6	0.172
2	0.106	7	0.093
				3	0.089	8	0.097
4	0.098	9	0.079
				5	0.185	10	0.107

Glycolic acid ethoxylate thing lauryl ether

Sample number into spectrum	Without PC	Sample number into spectrum	Containing PC
				1	0.295	6	0.297
2	0.584	7	0.107
				3	0.091	8	0.101
4	0.103	9	0.082
				5	0.202	10	0.115

Polyoxyethylene 2 oleyl ether

Sample number into spectrum	Without PC	Sample number into spectrum	Containing PC
				1	0.292	6	0.64
2	0.112	7	0.092
				3	0.097	8	0.1
4	0.096	9	0.08
				5	0.247	10	0.105

Lauryl sorbitan

Sample number into spectrum	Without PC	Sample number into spectrum	Containing PC
				1	0.396	6	0.106
2	0.104	7	0.091
				3	0.092	8	0.098
4	0.095	9	0.08
				5	0.169	10	0.105

Embodiment 7

It is applied in combination docusate sodium, Brij52 and S-PC to be incorporated in by the lysozyme of different protein concentrations In reverse micelle in mineral oil, and compared with when being used alone S-PC

1. construct, according to the mode described in embodiment 5, the hydrophobicity system utilizing S-PC as amphiphile Agent, difference is, adjusts the amount of lysozyme so that the final concentration of protein in mineral oil is the 50mg/ml shown in following table To 0.Lipid concentration is 100mg/ml.

2. construct according to the mode described in embodiment 5 and utilize docusate sodium, Brij52 and S-PC conduct The hydrophobic formulation of amphiphile, difference is, adjusts the amount of lysozyme so that final concentration of protein in mineral oil For the 50mg/ml shown in following table to 0.Lipid concentration is 100mg/ml.

3., after adding mineral oil, all samples is mixed on mixing roll mill 2 hours, then by 100 μ l's Every kind of sample is transferred in the single hole of clear microplate, measures the optical density at 620nm.Result such as following table institute Show.

Visible, for amphiphile mixture, until all achieving albumen in oil at concentrations up to 25mg/ml Clear solution (being indicated by the OD reading less than 0.2)；And in the preparation containing only SPC, only at egg White concentration could realize dissolving when being below 3.125mg/ml.Therefore, in terms of the amount of the albumen can being incorporated in oil, Method of the present invention is far superior to the method described in prior art (such as WO96/014871).

Embodiment 8

Be incorporated in mineral oil by lysozyme when being applied in combination docusate sodium, Brij52 and S-PC contains Have in the reverse micelle of amphiphile of varying level, and compared with when being used alone S-PC

1. construct, according to the mode described in embodiment 5, the hydrophobicity system utilizing S-PC as amphiphile Agent, difference is, adjust the amount of soybean lecithin so that the lipid final concentration of 100mg/ml in oil, 87.5mg/ml、75mg/ml、62.5mg/ml、50mg/ml、37.5mg/ml、25mg/ml、12.5mg/ml、 6.25mg/ml, 2.5mg/ml and 0mg/ml.After making lipid residue be completely dried, add enough mineral oil To realize the final volume (setting the density of amphiphile as 1g/ml) of 400 μ l.The final concentration of 1mg/ml of lysozyme in oil.

2. construct according to the mode described in embodiment 5 and utilize docusate sodium, Brij52 and S-PC conduct The hydrophobic formulation of amphiphile, difference is, adjusts the amount of soybean lecithin so that lipid is dense for the end in oil Degree for 100mg/ml, 87.5mg/ml, 75mg/ml, 62.5mg/ml, 50mg/ml, 37.5mg/ml, 25mg/ml, 12.5mg/ml, 6.25mg/ml, 2.5mg/ml and 0mg/ml.After making lipid residue be completely dried, add Enough mineral oil is to realize the final volume (setting the density of amphiphile as 1g/ml) of 400 μ l.Lysozyme in oil is eventually Concentration is 1mg/ml.

Visible, for amphiphile mixture, until concentration as little as 6.25mg/ml (amphiphile: protein ratio is 6.25:1 (w/w)) all achieve albumen clear solution (being indicated by the OD reading less than 0.2) in oil； And in the preparation containing only SPC, be only more than 100mg/ml (amphiphile: protein ratio in amphiphile concentration For 100:1 (w/w)) time could realize dissolve.Therefore, at the economic aspect of amphiphile demand, of the present invention Method be far superior to the method described in prior art (such as WO96/014871).

Embodiment 9

The manufacture of the vaccine product containing II Collagen Type VI

1. in the vial of 1 8ml band screw lid, add 0.5ml glacial acetic acid and 4.5ml dimethyl sulfoxide (DMSO), and vibrate be sufficiently mixed.This mixture of 1ml is added in the II Collagen Type VI of 5mg, then in room The lower gentleness mixing of temperature is overnight so that it dissolves.

2. in the vial of 3 8ml band screw lids, weigh docusate sodium, S-PC and Brij52 Each 200mg, is dissolved in while heating in 1.8ml hexamethylene.In a 8ml bottle, distribute 1.5ml's Many storehouses ester solution, the Brij52 solution of 1.5ml and the phospholipid solution of 1ml, and be sufficiently mixed.

3. the amphiphile of 1ml solution in hexamethylene is transferred in new 8ml vial, adds 0.2ml From the collagen solution of step 1, quick vortex mixed 30 seconds, then in-30 DEG C of cooling baths of glycerin/water under vibration Quick freezing.Bottle is placed on ice 5 minutes, be then transferred in-30 DEG C of refrigerators place 20 minutes.

4. make the content of bottle be exposed to the vacuum of 1mbar at+4 DEG C with Genevac vacuum pump, and overnight freeze Dry.After drying, adding the mineral oil of 900 μ l in vial content, gentle vibration is until all the elements thing all dissolves. Collagen concentration in oil is 1mg/ml.

5. weigh in 200ml glass spinner flask 20g gelatin, and while vibration, add 80g distilled water. Then mixture is heated on magnetic stirring apparatus at 50 DEG C, until all protein dissolutions.

6. 3ml gelatin solution is transferred in the 8ml vial of 6 pre-warms in 37 DEG C of water-baths.

7 add the 120 μ l oil (containing 120ug collagen) from step 4 in each bottle, and by slow vortex Mix and within 10 seconds, carry out gentle mixing.Bottle is made at room temperature to stand until content solidifies.After purging with nitrogen By bottle cover, be stored at+4 DEG C standby.The collagen of 10ug dosage is included in the gelatin solution of about 0.25ml In.

Embodiment 10

The bacterin preparation containing collagen is used to carry out the tight of the Orally administered rheumatoid arthritis lowering mouse model The embodiment of weight degree

1. the male DBA-1 mouse weights of pair 10～12 week old, and it is classified as shown below 3 group (n=8/ group). To all groups of all oral medications (passing through gavage) 4 times (the-10th day, the-7th day, the-5th day and the-3rd day), then exist The 100 μ g collagens that tail base portion is injected in complete Freund's adjuvant, thus induce collagen-induced arthritis (CIA).

2., at the 0th day all induced arthritis in all animals, strengthened at the 21st day subsequently, strengthened 5 days, the 7th day, the 9th day, the 12nd day, the 14th day, the 16th day, the 19th day and the 21st day are to sickness rate and serious journey Degree is estimated and marks.3 treatment groups (often 8 animals of group) are used as described below:

I () .CIA+ only raises by force with oil

(ii) .CIA+ oil containing cattle II Collagen Type VI (10 μ g/ agent) is raised by force

(iii) .CIA+ cattle II Collagen Type VI (20 μ g/ agent) is raised by force

During testing, after carrying out CIA induction, arthritic rate (is often suffered from group in any amount of joint The % of the arthritic animal of any order of severity) and arthritis severity (the serious journey of disease in every individual mice Degree) carry out clinical score.Use following standard arthritis scoring system.For every animal, evaluate every pawl Swelling and deformation, carry out marking and calculating summation.

0 point: without arthritis

1 point: only 1～2 toe is impacted

More than 2 points: 3 toes, and/or pawl swelling (metacarpal bone/rear shank)

3 points: carpopodium/shank swelling

4 points: deformation and carpopodium/midtarsal joints are tetanic

3., when research terminates (the 21st day), make mice euthanasia, before and after collection lower limb and 10% neutral buffered Formalin is fixed, 10% formic acid in 5% formalin carries out decalcification, and carries out paraffin embedding.By the right side Kneed sagittate section is with toluidine blue and fast green dyeing.Utilize standard histopathologic rating system (see annex 1) By single blind observer (CBL), tangent plane is marked.

Such as finding in fig 1 and 2, observe with when being used alone 20 μ g collagen compared with, for 10 μ g in oil The collagen of dosage, it was observed that suffer from the bigger minimizing of arthritic size of animal.It addition, the effect to morphological change Really.

Claims

1. a single-phase hydrophobic formulation, described preparation comprises the hydrophilic species in oil phase and amphipathic component, institute State amphipathic component and comprise docusate sodium, phospholipid and nonionic amphiphile, wherein, described hydrophilic species part quilt Described amphipathic component is surrounded, and the hydrophilic head group of described amphipathic component is towards described hydrophilic species, And wherein, between described amphipathic component and described hydrophilic species, there is no chemical interaction；Described preparation Being characterised by, described nonionic amphiphile has: the lipophilic chain comprising 10 to 20 carbon, and comprise 2 to 10 ethyleneoxy group or the head group of 1 to 3 hydroxyl.

2. preparation as claimed in claim 1, wherein, described hydrophilic species are selected from: peptide, albumen, lipid, Sugar, nucleic acid, steroid, and/or the conjugate that the combination of one or more these reagent is formed, and/or with at least Article one, middle chain or the conjugate of long chain hydrocarbon afterbody.

3. the method manufacturing the hydrophobic formulation containing hydrophilic species, said method comprising the steps of:

I () will be dissolved in the solution of the docusate sodium of hydrophobic solvent, phospholipid and nonionic amphiphile and be soluble in the aqueous phase Hydrophilic species solution mixing, to form emulsion；

(ii) described aqueous phase and hydrophobic solvent are removed；

(iii) in the residue being dried obtained in (ii), oil phase is added；

Wherein, between described docusate sodium, phospholipid and nonionic amphiphile and described hydrophilic species, do not has chemistry Interact；Described nonionic amphiphile has: the lipophilic chain comprising 10 to 20 carbon, and comprise 2 to 10 ethyleneoxy group or the head group of 1 to 3 hydroxyl.

4. method as claimed in claim 3, wherein, described hydrophobic solvent is selected from hexamethylene, cycloheptane, ring Octane or its mixture and the optional tert-butyl alcohol.

5. the method as described in claim 3 or 4, wherein, step (ii) is carried out by lyophilizing.

6. the method as described in claim 3 or 4, wherein, described hydrophilic species are selected from: peptide, albumen, fat Matter, sugar, nucleic acid, steroid, and/or the conjugate that the combination of one or more these reagent is formed, and/or with Chain or the conjugate of long chain hydrocarbon afterbody at least one.

7. the method as described in claim 3 or 4, wherein, described hydrophilic species are collagen or its fragment, institute State in the mixture that hydrophilic species are dissolved in acetic acid and dimethyl sulfoxide.

8. a two-phase compositions, described compositions comprises: aqueous phase；With described in claim 1 or 2 single-phase dredge Aqueous formulation or the single-phase hydrophobic formulation that can obtain by the method according to any one of claim 3 to 7.

9. two-phase compositions as claimed in claim 8, wherein, described two-phase compositions is O/w emulsion.

10. two-phase compositions as claimed in claim 8 or 9, wherein, described aqueous phase comprises gelatin or albumin.

11. two-phase compositions as claimed in claim 10, wherein, described two-phase compositions is microcapsule.

12. 1 kinds of methods forming two-phase compositions, described method includes: make the preparation described in claim 1 or 2 Or can contact with aqueous solution with the preparation that the method according to any one of claim 3 to 7 obtains.

13. methods as claimed in claim 12, wherein, described aqueous solution comprises gelatin or albumin.

Any one of 14. preparations described in claim 1 or 2 being used for medical applications or claim 8 to 11 Described compositions, maybe can be with the preparation that the method according to any one of claim 3 to 7 obtains, maybe can use The compositions that method described in claim 12 or 13 obtains.

15. 1 kinds of compositionss, described compositions comprises: the preparation described in claim 1 or 2 or claim 8 To the compositions according to any one of 11, maybe the method according to any one of claim 3 to 7 can be used to obtain Preparation, the compositions that maybe can obtain by the method described in claim 12 or 13.

16. compositionss as claimed in claim 15, described compositions also comprise one or more drug excipients, Diluent or supporting agent.

17. compositionss as described in claim 15 or 16, described compositions is suitable to Orally administered, intramuscular and uses Or subcutaneous administration.

18. compositionss as described in claim 15 or 16, described compositions comprises collagen, is used for treating rheumatoid Property arthritis.

19. 1 kinds of vaccines, described vaccine comprises described in the preparation described in claim 14 or claim 15 or 16 Compositions.

20. vaccines as claimed in claim 19, described vaccine is suitable for Orally administered.

21. vaccines as claimed in claim 20, described vaccine includes capsule.

22. vaccines as claimed in claim 21, wherein, described capsule has enteric coating.

23. vaccines as according to any one of claim 19 to 22, wherein, described compositions comprises malaria antigen Or influenza antigens or intestinal tract disease pathogen antigen.

24. 1 kinds of capsules with enteric coating, described capsule comprises the compositions described in claim 15 or 16.

25. 1 kinds of cosmetic preparations, described goods comprise: the preparation described in claim 1 or 2 or claim Compositions according to any one of 8 to 11, maybe can obtain by the method according to any one of claim 3 to 7 Preparation, maybe can with described in claim 12 or 13 method obtain compositions.

26. cosmetic preparations as claimed in claim 25, described goods also comprise one or more excipient, dilute Release agent or supporting agent.